The ligand-dependent transcription factor ROR?t regulates the development and functions of lymphocytes, including Th17 cells and type 3 innate lymphoid cells (ILC3) that have central roles in mucosal barrier protection and in mediating multiple autoimmune inflammatory diseases. Targeting of ROR?t by small molecule inhibitors is a strategy that has been successfully validated in animal models and is being developed for therapeutic application in human autoimmune diseases, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and, potentially, multiple sclerosis. Although many of the targets of ROR?t and other key transcription factors involved in Th17 cell differentiation have been identified, the roles of ROR?t in the gene regulatory programs of other cell types, e.g. thymocytes, ILC3, and lymphoid tissue inducer cells, have not been studied, nor have the roles of the closely-related ROR? isoform that is expressed outside of the immune system. A better understanding of how ROR?t executes cell type-specific functions may facilitate development of better strategies to selectively inhibit its activities in the desired cells and tissues. We have ued genetic and proteomics screens to identify molecules that contribute to ROR?t-dependent target gene regulation in Th17 cells, either indirectly or directly by associating with ROR?t complexes. Molecules identified in both screens include nuclear pore proteins that have been recently implicated as having roles in the nucleoplasm as well as the nuclear envelope. We propose to study the roles of Nup98 and Nup153, proteins that interact with ROR?t and contribute to the expression of a large number of ROR?t target genes. In preliminary studies, we have identified mutations in ROR?t that abrogate interaction with Nup153. Mice with one of these mutations have impaired Th17 cell induction but normal T cell development. We will extend our studies with Th17 cells, and also study the role of the ROR?t-nucleoporin interactions in differentiation and function of TCR-??-17 cells and ILC3 and in the development of lymphoid tissue inducer cells and thymocytes. For the first aim, we will characterize the biochemical basis of the interaction of ROR?t with Nup153 and Nup98 and will determine how this interaction influences formation of ROR?t transcriptional complexes and the DNA occupancy of each factor in polarized Th17 cells. We will also examine the effect of ligand binding on association of ROR?t with its partners, taking advantage of small molecule antagonists and our finding that Nup98 regulates enzymes involved in cholesterol biosynthesis, which may thus influence endogenous ligand availability. In Aim 2, we will introduce relevant mutations into the mouse germ line and examine the effect on chromatin accessibility and gene expression programs in Th17 cells generated in vitro and in vivo and in other ROR?t-dependent cells. In Aim 3, we will use mice with the various point mutations in ROR?t or its partner proteins to determine the effects in models of Th17-mediated autoimmune disease or ILC3-dependent barrier protection. Together, these studies will help in the design of better screens for cell type-selective targeting of ROR?t functions.